Theasaponin E1 destroys the salt tolerance of yeasts

Cells of Zygosaccharomyces rouxii in a medium containing a high concentration of NaCl were killed during incubation for 2–4 h with a low concentration of a mixture of saponins from tea seeds (TSS). The higher the concentration of NaCl in the medium, the higher the inhibitory effect of TSS on the growth of the yeast. The above inhibitory effect of TSS on the growth of the yeast was not observed when cells were incubated in hypertonic media composed of nonionic substances such as sugars. The ATPase activity of plasma membrane preparations from the yeast cells was slightly affected by the addition of TSS. It is shown that TSS facilitates leakage of glycerol from the yeast cells under NaCl-hypertonic conditions. The major inhibitor in the mixture of saponins was isolated and identified as theasaponin E₁. Its isomer, theasaponin E₂, did not have any effect on the salt tolerance of Z. rouxii or Saccharomyces cerevisiae.     

Salt shock enhances the expression of ZrATP2, the gene for the mitochondrial ATPase beta subunit of Zygosaccharomyces rouxii

In the course of a study of cell wall proteins from the salt-tolerant yeast Zygosaccharomyces rouxii, a protein that increased its expression as the NaCl concentration of the culture medium increased was identified. Several degenerate primers were constructed based on partial amino acid sequences of this protein and were used in PCR amplification of a gene termed ZrATP2. The amino acid sequence deduced from nucleotide sequence of the gene revealed that ZrATP2 encodes the beta subunit of mitochondrial F1 ATPase. Northern blot analysis demonstrated that NaCl shock induced an elevation in ZrATP2 expression, which corresponded with the resumption of Z. rouxii cell growth after salt shock.     

2,5-Dimethyl-4-hydroxy-3(2H)-furanone as a secondary metabolite from D-fructose-1,6-diphosphate metabolism by Zygosaccharomyces rouxii

2,5-Dimethyl-4-hydroxy-3(2H)-furanone (DMHF) is an important aroma compound found in many fruits such as strawberries and pineapples and it is also produced by the soy-sauce-fermenting yeast Zygosaccharomyces rouxii after the addition of d-fructose-1,6-diphosphate to yeast-peptone-dextrose nutrient media. Dilute DMHF solutions exhibit a strawberry-like flavor while DMHF concentrates have a caramel-like aroma. In media containing D-fructose-1,6-diphosphate as the sole carbon source, growth of Z. rouxii and formation of DMHF were not observed. Although Z. rouxii cells grew in media with D-glucose as the sole carbon source, DMHF was only produced when media were supplemented with D-fructose-1,6-diphosphate. The DMHF concentration always correlated with the yeast cell count and D-fructose-1,6-diphosphate concentration. Addition of CaCl2 (up to 50 g.l(-1)) led to a higher DMHF concentration. Addition of Na2SO3 reduced the growth of Z. rouxii and inhibited DMHF formation. The amount of DMHF formed by Z. rouxii was not significantly affected by the addition of KH2PO4. DMHF concentrations of 5 and 10 g.l(-1) partially and completely inhibited the growth of Z. rouxii cells, respectively. Only the singly labeled furanone was formed after the addition of 1-13C-D-fructose-1,6-diphosphate to the medium. However, unlabeled DMHF was formed in the presence of (13)C(6)-D-glucose. Therefore, the carbons of the furanone originate exclusively from exogenously supplied D-fructose-1,6-diphosphate as no exchange with the internal pool of D-fructose-1,6-diphosphate occurs. This implies that DMHF is a secondary metabolite of Z. rouxii formed from D-fructose-1,6-diphosphate. We assume that at least the first step of the metabolism of D-fructose-1,6-diphosphate takes place in the cell wall or membrane of the yeast.     

甘油与鲁氏酵母共固定在高盐稀态酱油酿造中的应用

本文旨在研究相容性溶质之一的甘油对高渗透压环境下鲁氏结合酵母的保护作用,以及将甘油与鲁氏结合酵母共固定对高盐稀态酱油发酵的影响。添加不同浓度甘油至含3 mol/L氯化钠的YPD培养基中,观察酵母菌生长情况。同时,共固定甘油与鲁氏结合酵母,应用于高盐稀态酱醪发酵。结果显示,1.37 mmol/L甘油即可有效缓解高盐环境对鲁氏结合酵母的生长抑制作用,生长延滞期缩短达4~6 h。高盐稀态酱油发酵中试结果显示,与空白对照组相比,试验组蛋白质转化率提高幅度达2.23~5.64%,头油中各理化指标平均提前15 d达到空白组相同水平。综上所述,甘油作为一种相容性溶质可有效保护高盐环境下鲁氏结合酵母的生长繁殖,及其在高盐稀态酱醪发酵中的代谢性能,甘油与鲁氏接合酵母共固定技术能够提高酱油发酵的原料利用率、缩短发酵周期,在酱油酿造领域具有一定的实际应用价值。     

生香酵母筛选鉴定及其对辣椒酱发酵的影响

为了获得适用于辣椒酱发酵的生香酵母,从自然发酵辣椒酱中筛选优良菌种,并对菌种形态特征、生理生化特性、菌种发酵辣椒基料等进行了研究。经筛选,获得一株生香能力较强的菌株LA-Y09,该菌株被鉴定为鲁氏酵母（Saccharomyces rouxii）。菌种LA-Y09的最适生长温度为28 ℃,在NaCl质量分数为12%的培养基中仍有较高菌数,说明其具有良好的耐盐特性,在质量分数为8%的辣椒基料中能够生成多种呈现蜜香和果香的香气成分,使辣椒发酵醪香气得到明显提升。     

一株耐盐产香鲁氏接合酵母FA-1的鉴定及其在酱油酿造中的应用

为提高酱油风味与品质,从酱油醪液中筛选耐盐产香酵母菌株并应用于酱油酿造。对筛选菌株进行鉴定,并采用顶空固相微萃取-气质联用法（HS-SPME-GC-MS）分别检测其在不同NaCl浓度的发酵液及酱油成品中的挥发性香气成分。结果表明,筛选出1株耐盐产香菌株FA-1,经鉴定为鲁氏接合酵母（Zygosaccharomyces rouxii）,可耐受22%的食盐浓度。在不同NaCl浓度的酱油发酵液中共检出56种挥发性风味物质,且当NaCl质量分数为18%时,更有利于醇类化合物的富集。在酱油生产试验中,添加鲁氏接合酵母FA-1的实验组中醇类化合物占比最大（72.03%）,其中苯乙醇、3-甲基-1-丁醇、乙醇的含量较高,且醇类和酯类化合物的含量分别较添加酵母As 1.039的对照组提高131.05%和189.65%。因此,菌株FA-1具有良好的耐盐产香性能,可作为潜在的酱油发酵菌株。     

高盐稀态酱油发酵S3—2酵母基础发酵性能的研究

鲁氏酵母（S酵母）的耐盐能力抑制了其对高盐稀态酱油风味的改善作用。S3-2酵母是本试验室以S酵母为出发菌株,利用基因组重组技术构建的一株具有高耐盐度的酵母菌株。在YPD发酵培养基中对S3-2酵母进行基础发酵性能研究的结果表明,S3-2比S酵母具有更高的耐盐能力,S酵母能生长的最大盐度为19％,而S3-2酵母在含盐量高达21％的培养基中仍能够良好生长。在含盐量为18％的YPD发酵培养基中,S3-2酵母在提高醇类物质的种类和含量,对提高高盐稀态酱油风味具有重要的意义。     

Sodium chloride decreases the bacteriocidal effect of acid pH on Escherichia coli O157:H45

To reduce the risk of foodborne illness, many fermented and minimally processed foods rely on the "hurdle effect", i.e. a combination of two or more inhibitory agents being more inhibitory than any of the agents alone. However, we have observed that such a combination of agents is not always more inhibitory to foodborne pathogens than one alone. In this paper, we show that a combination of NaCl and acid pH is less effective than acid pH alone in reducing the numbers of Escherichia coli O157:H45. The presence of sodium chloride reduces the bacteriocidal effect of lactic acid on logarithmically growing cells of E. coli O157:H45. with approximately 10(3)-fold more survivors at pH(o) 4.2 when 4% NaCl was added to the medium. A similar protective effect was also seen with other organic acidulants and E. coli strains. The cytoplasmic pH (pH(i)) of cells in medium at pH 4.2 containing added salt was 5.8 which was 0.56 units higher than that of cells in the same medium without added salt. When the pH(i) of cells in medium without added salt was adjusted to the same value (5.8) by adding KOH, the rate of survival was also considerably greater than that of cells in medium without added salt. These data suggest that E. coli can use NaCl to counteract acidification of its cytoplasm by organic acids, and in addition, that combinations of antimicrobial agents cannot always be relied upon to achieve additive antimicrobial effects.     

硫胺素对鲁氏接合酵母高盐适应性的影响

采用同位素内标-超高效液相色谱串联三重四极杆质谱（UPLC-MS/MS）法测定金沙江攀枝花江段6种常见鱼中卡巴氧代谢物喹恶啉-2-羧酸（QCA）和喹乙醇代谢物3-甲基-喹恶啉-2-羧酸（MQCA）的残留量。样品经过预处理,采用ZORBAX SB-C18色谱柱分离,以0.1%甲酸水-乙腈为流动相进行梯度洗脱;采用电喷雾-正离子多反应监测模式（MRM）检测,同位素内标法定量。结果表明,QCA和MQCA的质量浓度为2.0～100.0 ng/mL时,线性关系良好（R＞0.999）,回收率为91.4%～100.4%,精密度试验结果相对标准偏差（RSD）为6.3%～11.5%,定量限为0.2 μg/kg,检出限为0.05 μg/kg。该方法灵敏度及准确度良好,能满足6种常见鱼的组织中卡巴氧及喹乙醇代谢物残留分析的要求。     

The effect of 100% CO2 on the growth of nonproteolytic Clostridium botulinum at chill temperatures

The growth of a cocktail of spores from six nonproteolytic Clostridium botulinum type B and E isolates at 5 and 10 degrees C was used to assess the combined effect of NaCl (0.5-4.5% w/v), pH (5.5-6.5) and atmosphere (10% H2:90% N2, 5% CO2:10% H2:85% N2, or 100% CO2) in buffered peptone, yeast, glucose, starch broth with an Eh of approximately -350 mV. Under all atmospheres growth tended to be slower as the concentration of NaCl increased and with NaCl combined with pH levels below 6.0. Of the atmospheres tested, growth occurred at a slower rate and over a narrower range of conditions when C. botulinum was exposed to 100% CO2. This effect was enhanced when the incubation temperature was 5 degrees C. The results indicate that while CO2 decreased C. botulinum growth at chill temperatures, prevention of growth also depended on the NaCl concentration and the pH of the medium.     

酵母对无机锌的富集及其影响因素研究

在培养基中添加无机锌，利用生物转化技术，通过酵母的生命活动，将无机锌有效地富集到细胞内，研究锌离子浓度对酵母生长繁殖、细胞含锌量、锌富集率及酵母发酵力的影响，并通过控制锌离子浓度，使酵母细胞既能富集锌，又对其发酵力不产生显著的影响，以便将仍具有发酵活力的富锌酵母用于食品加工之中。研究结果表明，锌离子浓度对酵母的生长繁殖和富集能力影响较大，浓度越高，酵母细胞含锌量越大。但无机锌对酵母细胞有显著的损伤，且这种损伤是非遗传性的，当无机锌去掉后，酵母细胞形态又恢复正常。     

The effect of inoculum size on the lag phase of Listeria monocytogenes.

The effect of inoculum size on population lag times of Listeria monocytogenes was investigated using the Bioscreen automated microtitre plate incubator and reader. Under optimum conditions, lag times were little affected by inoculum size and there was little variation between replicate inocula even at very low cell numbers. However, in media containing inhibitory concentrations of NaCl, both the mean lag time and variation between replicate inocula increased as the inoculum size became smaller. The variation in lag time of cells within a population was investigated in more detail by measuring the distribution of detection times from 64 replicate inocula containing only one or two cells capable of initiating growth. The variance of the lag time distribution increased with increasing salt concentration and was greater in exponential than in stationary phase inocula. The number of cells required to initiate growth increased from one cell under optimum conditions to 10(5) cells in medium with 1.8 M NaCl. The addition of spent medium from a stationary phase culture reduced the variance and decreased lag times. The ability to initiate growth under severe salt stress appears to depend on the presence of a resistant sub-fraction of the population, although high cell densities assist adaptation of those resistant cells to the unfavourable growth conditions by some unspecified medium conditioning effect. These results are relevant to the prediction of lag times and probability of growth from low numbers of stressed cells in food.     

山苍子油对花生酱的防霉保鲜效果

采用平板法比较山苍子油与合成食品防腐剂对５种霉菌包括引起花生酱变质的霉菌的抗菌效力。结果表明：在培养基ｐＨ５．５时，山苍子油与对多数霉菌的最低抑制浓度为１．７７ｍｇ／ｍｌ，而山梨酸钾为２．２１ｍｇ／ｍｌ，苯甲酸钠为３．１０ｍｇ／ｍｌ。同时，从山苍子油与黄曲霉产毒关系的试验中发现，山苍子油对贡曲霉产生黄曲霉毒素有较强的抑制作用。在鲁氏酵母的麦芽汁摇床培养中，添加０．１０％的山苍子油能改变及生长曲线，     

油茶粕中茶皂素纯化方法与抗菌活性研究

对Amberliter XAD-16和DM130大孔树脂纯化油茶皂素的条件进行探索,比较两种树脂纯化的油茶皂素和AB-8大孔树脂纯化产品的纯度,并测定纯化产品与粗提物的抗菌效果。结果表明,在相应的最佳条件下,Amberliter XAD-16纯化所得的茶皂素产品纯度低,为74.5%;DM130大孔树脂处理的高,为85.6%;AB-8大孔树脂居中,为83.5%。油茶皂素的粗提物和纯化物对供试微生物的抗菌效果一致,纯化物抑制程度略高;二者均对玉米纹枯病菌抑制效果最明显,对白色葡萄球菌、白色念珠菌和腊状芽孢杆菌的抑菌效果次之,对温和气单胞菌、豚鼠气单胞菌、嗜水气单胞菌、变形杆菌的抑制效果不明显,对黑曲霉、米曲霉的生长没有抑制作用。     

丁香和肉桂精油气相抑菌活性研究

采用超临界CO2萃取法提取丁香和肉桂精油,并通过滤纸片法、固相扩散法、气相扩散法等体外抗菌试验,比较了丁香、肉桂精油及混合精油和常用化学防腐剂山梨酸钾对11株食品中常见污染菌的抑菌作用。结果表明:丁香、肉桂精油及混合精油对供试细菌、霉菌和酵母菌均有较强的抑制作用,抑菌效果显著优于山梨酸钾;对霉菌的抑菌作用显著高于酵母菌和细菌,且气态抑制作用比固相抑菌作用强;混合精油对细菌的抑菌作用有拮抗作用,在气相中的拮抗作用更强;对酵母菌的抑菌作用没有影响;对黄曲霉和黑曲霉有相加作用;对桔青霉有协同作用。     

Impact of sodium chloride on Escherichia coli O157:H7 and Staphylococcus aureus analysed using transmission electron microscopy

Abundant literature information is available on sodium chloride, NaCl, as an antimicrobial and a preservative, however, information on NaCl effects on bacterial cell morphology is lacking. The effect of NaCl, on Escherichia coli O157:H7 and Staphylococcus aureus cells individually grown in a laboratory medium was examined using transmission electron microscopy (TEM). Cultures were grown in brain heart infusion (BHI) broth containing dissolved 0%, 5%, or 10% (w/v) commercially obtained fine (FN) and extra coarse (EC) grade granular NaCl. The pathogens were incubated at 35 degrees C for 12 and 24 h. Then, a mixture of five strains of each pathogen per treatment was prepared. Samples were centrifuged, pellets collected, fixed immediately with glutaraldehyde, and prepared for TEM examination. Cells morphology on TEM micrographs verified that the magnitude of morphological damage to E. coli O157:H7 cells was significantly greater than that of S. aureus cells. More cell injury occurred as NaCl concentration increased from 5% to 10%. Generally, S. aureus maintained its cellular structure and no severe cell wall or plasma membrane damage and/or shrinkage was observed. At 10% NaCl, the damage to E. coli O157:H7 cells was extensive, and the pathogen seemed to have lost its cellular integrity. Although NaCl affected the morphology of E. coli O157:H7 and S. aureus, the coarse grade of NaCl seemed to have a milder effect with respect to cell damage, especially on S. aureus. The 24 h-old cultures were more susceptible to NaCl treatment compared to the 12 h-old cells. Thus, the age of the cells has an impact on their resistance to salt--the environmental stressor.     

Survival of staphylococcus aureus and Escherichia coli as affected by ethanol and NaCl

Growth of three strains of Staphylococcus aureus and two strains of Escherichia coli on nutrient agar (NA) supplemented with ethanol and NaCl was investigated. S. aureus did not grow on NA containing > or =10% ethanol (wt/wt) combined with > or =0% NaCl (wt/wt), or 7.5% ethanol combined with 7.5% NaCl. Neither E. coli nor E. coli O157:H7 grew on NA containing > or =7.5% ethanol combined with > or =0% NaCl, 5% ethanol combined with > or =2.5% NaCl, or > or =5% NaCl combined with > or =0% ethanol. It is apparent that NaCl enhanced the inhibitory effect of ethanol on growth of S. aureus and E. coli When cells were suspended in nutrient broth containing 12.5, 20, or 40% ethanol combined with NaCl, viable cells decreased with an increase of ethanol concentration. Ethanol sensitivity among strains and between genera varied in a limited range. When the cells were exposed to 20% ethanol in combination with 5% NaCl, S. aureus and E. coli lost viability after 30 and 10 min, respectively. When treated with 40% ethanol combined with > or =0% NaCl, all test strains lost viability within 5 min.     

Effect of heterologous expression of molecular chaperone DnaK from Tetragenococcus halophilus on salinity adaptation of Escherichia coli

Molecular chaperone DnaK of halophilic Tetragenococcus halophilus JCM5888 was characterized under salinity conditions both in vitro and in vivo. The dnaK gene was cloned into an expression vector and transformed into Escherichia coli. The DnaK protein obtained from the recombinant E. coli showed a significantly higher refolding activity of denatured lactate dehydrogenase than that from non-halophilic Lactococcus lactis under NaCl concentrations higher than 1 M. E. coli without the overexpression of DnaK exhibited a growth profile with a prolonged lag phase and suppressed maximum cell density in Luria-Bertani medium containing 5% (0.86 M) NaCl. On the contrary, the overexpression of T. halophilus DnaK greatly shortened this prolonged lag phase with no effect on maximum growth, while that of L. lactis DnaK decreased maximum growth. The amount of protein aggregates was increased by salt stress in the E. coli cells, while this aggregation was greatly suppressed by the overexpression of T, halophilus DnaK. These results suggest that heterologous overexpression of T. halophilus DnaK, via its chaperone activity, promotes salinity adaptation of E. coli.     

牛胎盘废弃物制备食品微生物培养基

采用废弃牛胎盘下脚料为氮源制备新型培养基。并采用Plackett-Burman法、最陡爬坡试验和响应面试验(Box-Behnken设计法)相结合的方法对大肠杆菌培养基进行优化。结果表明:牛胎盘下脚料经水解后,替代LB培养基中的酵母浸粉,显示出较高的生长水平。优化后的新型培养基(NTP)组成为:葡萄糖2.8 g/L,NaCl 11 g/L,胰蛋白胨10.3 g/L,牛胎盘水解物16.7 g/L,pH 7.85,最终得到活菌数量较高的培养基配方。     

Inhibitory Effect of Fusarium Mycotoxins on Growth of Brewing Yeasts. 2. Deoxynivalenol and Nivalenol*

The effect of the trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) on growth of Saccharomyces cerevisiae lager and ale strains has been studied. The toxins were added into the growth medium in low and high concentrations. Yeast growth was assessed by measurement of dry weight or relative growth, cell number, viability and conductance change of the growth medium using direct and indirect methods. The inhibitory effect of both DON and NIV on yeast growth was dependent on toxin concentration. Additionally, when the extent of inhibition of yeast growth caused by high concentrations of both toxins was observed, it was subject to yeast strain, length of incubation and method used to assess yeast growth. The lowest concentrations of mycotoxin causing significant inhibition on growth of brewing yeasts were: 100 μg/ml DON for the lager strain and 50 μg/ml for the ale strain, and 50 μg/ml NIV for the ale strain.